Class switch recombination (CSR) and somatic hypermutation (SHM) are two pivotal processes in the immune system and in lymphoid leukemias and lymphomas. Activation-induced deaminase (AID) initiates both CSR and SHM by deaminating C to U. However, AID can only act on single- stranded DNA (ssDNA). How is AID directed to the switch recombination regions for CSR and to the VDJ or VJ segments for SHM? More than one process may help target AID. For CSR, the highly distinctive G-richness of the nontemplate DNA strand of all mammalian switch regions causes R-loop formation when an RNA polymerase passes through these regions. This provides one important level of AID targeting, including the ssDNA required by AID. The rules governing R-loop formation in vitro using prokaryotic RNA polymerases have been defined on this grant in the 2005-2009 cycle. Using an in vivo functional assay, we are now in a position to determine if these rules apply within living mammalian cells. Once established, the rules for R-loop formation will be key in determining the contribution of R-loop formation at sites of rearrangement or instability elsewhere in the genome. This proposal has four aims that define key rules for in vivo R-loop formation. In Aim 1, we test whether a nick can potentiate R-loop formation in vivo, as it does in vitro. If so, then it would explain R-loop formation and CSR upstream of switch repetitive zones, and it would suggest a mechanism by which short R-loops might form for SHM at V(D)J regions, as tested for in Aim 4. Moreover, it would explain how R-loop formation might occur much more readily at many sites in the genome. In Aim 2, S1 knockin alleles will be varied to test whether G-clusters are critical for R-loop initiation zones (designated RIZ), which are regions where R-loops consistently begin. We will also test if the length of the R-loop elongation zone (REZ) is determined by G-density. These fundamental features are key for us to predict where we can expect R-loops to form in the genome. In Aim 3, we test whether interfering with the major RNase H activity can affect CSR. Loss or reduction of RNase H would be expected to reduce ssDNA on the template DNA strand. Therefore, this aims seeks to reveal how AID exposes ssDNA on the template strand. In Aim 4, we use a mouse that is naturally monoclonal (via B cell nuclear transfer) at the IgH locus with a VDJ configuration to test for short R-loop formation as the basis for ssDNA within the regions that undergo SHM. These aims will provide insight into the rules and parameters that determine where R-loops form, determine how R-loops target AID in CSR and SHM, and help understand the roles of R-loops elsewhere in the genome in pathological or physiological states. PUBLIC HEALTH RELEVANCE: The ability of our bodies to send antibodies to the lungs, GI tract, and urogenital system requires a process in the immune system called class switch recombination (CSR). The ability of our immune system to mount a booster-shot response in order to generate effective antibodies against microorganisms relies on a process called somatic hypermutation (SHM). This grant is directed at understanding how these processes normally function and how they go awry in malignancy.